BALB.NCT-Cpoxnct is a unique mouse model of hereditary coproporphyria

In humans, mutations in the coproporphyrinogen oxidase (CPOX) gene can result in hereditary coproporphyria (HCP), characterized by high levels of coproporphyrin excretion in the urine and feces, as well as acute neurovisceral and chronic cutaneous manifestations. Appropriate animal models for comprehending the precise pathogenesis mechanism of HCP have not been reported that show similarities in terms of gene mutation, reduced CPOX activity, excess coproporphyrin accumulation, and clinical symptoms. As previously discovered, the BALB.NCT-Cpoxnct mouse carries a hypomorphic mutation in the Cpox gene. Due to the mutation, BALB.NCT-Cpoxnct had a drastic increase in coproporphyrin in the blood and liver persistently from a young age. In this study, we found that BALB.NCT-Cpoxnct mice manifested HCP symptoms. Similar to HCP patients, BALB.NCT-Cpoxnct excreted an excessive amount of coproporphyrin and porphyrin precursors in the urine and displayed neuromuscular symptoms, such as a lack of grip strength and impaired motor coordination. Male BALB.NCT-Cpoxnct had nonalcoholic steatohepatitis (NASH)-like liver pathology and sclerodermatous skin pathology. A portion of male mice had liver tumors as well, whereas female BALB.NCT-Cpoxnct lacked these hepatic and cutaneous pathologies. In addition, we discovered that BALB.NCT-Cpoxnct exhibited microcytic anemia. These results indicate that BALB.NCT-Cpoxnct mice serve as the suitable animal model to help gain insight into the pathogenesis and therapy of HCP.


Introduction
Coproporphyrinogen oxidase (CPOX) is an enzyme that catalyzes the conversion of coproporphyrinogen III to protoporphyrinogen IX in the heme biosynthesis pathway. In humans, mutations in the CPOX gene that diminish enzyme activity can be a cause of hereditary coproporphyria (HCP; OMIM #121300), a genetically dominant disease with incomplete penetrance [1]. >65 CPOX mutations have been identified in HCP patients [1]. The majority of the HCP patients were heterozygous for the mutant and normal CPOX alleles. However, the presence of a mutant gene is insufficient for disease development, and most carriers of a disease-causing CPOX mutation do not exhibit clinical symptoms. HCP symptoms typically manifest in the 20s or 30s, with acute attacks triggered by factors, such as fasting, alcohol, sulphonamide antibiotics, and hormones such as progesterone [2]. Increased demand for hepatic heme synthesis is thought to be the mechanism by which these factors precipitate acute attacks. Reduced CPOX activity in a CPOX mutation carrier causes an accumulation of coproporphyrinogen III in tissues, part of which is then autoxidized to coproporphyrin III and excreted in excess in the urine and feces. Thus, excess excretion of coproporphyrin (exceeding three times the average level) is a crucial diagnostic indicator for HCP. HCP patients exhibit clinical symptoms of both hepatic and cutaneous porphyrias. Like hepatic porphyrias, HCP manifests clinically as acute neurovisceral attacks frequently accompanied by nausea, vomiting, and constipation. The additional neurovisceral manifestations are tachycardia, hypertension, motor weakness, and seizures. Acute attacks are reversible with prompt treatment such as haem arginate infusions that reduce the overproduction of δ-aminolevulinic acid (δ-ALA) [3][4][5]. Despite this, patients experience repeated acute attacks and remissions. Also, several papers have reported HCP patients with hepatocellular carcinoma [6][7][8][9][10]. In the Swedish porphyria register, the fraction of HCP patients that had primary liver cancer was 1/56 [10]. Around twenty to 30% of affected patients also develop skin lesions in the sun-exposed areas of the skin. Like cutaneous porphyrias, affected individuals may experience severe pain, burning, and itching of such areas (photosensitivity). Eventually, the skin may become fragile and develop blisters with fluid. These neurovisceral and dermal findings vary significantly among patients. The precise pathogenesis mechanism of HCP is not understood.
Animal models with appropriate Cpox mutations are essential for comprehending the precise pathogenesis mechanism of HCP. N-ethyl-Nnitrosourea (ENU) mutagenesis has produced two strains of Cpox knockout mice (RBC16 and M100835) [11,12]. A nonsense nucleotide substitution produces a premature stop codon in place of tryptophan at amino acid 373 in one strain of RBC16 [11]. Homozygotes for this mutant allele are prenatally lethal due to the complete shutdown of heme biosynthesis, whereas heterozygotes were born alive. The amount of CPOX in the livers of heterozygotes was sustained at around 50% of that of wild-type mice. Intriguingly, female heterozygous RBC16 mice excreted three-to fourfold higher levels of coproporphyrin in their urine and feces than wild-type female mice. No other HCP symptoms or notable histopathology were observed in RBC16 mice of both sexes. The lack of pathology and only a moderate increase in coproporphyrin in the urine and feces of female RBC16 mice is likely because the remaining CPOX activity is sufficient to prevent coproporphyrin accumulation in the cells. Another ENU mutagenized mouse strain, M100835, has a point mutation at the acceptor site of Cpox intron 4 that induced an abnormal splicing event [12]. Homozygotes for the mutant allele died in utero. The HCP phenotypes of the M100835 mice have not been studied. Thus, these Cpox knockout mice are incompetent to be used for the exploration of the pathological mechanisms of HCP. Meanwhile, mutant Cpox heterozygotes exhibited microcytic anemia, caused by heme deficiency due to decreased CPOX activity [11,12].
In a previous study, we determined hereditary cataracts in the BALB. NCT-Cpox nct is caused by a spontaneous missense mutation in the gene for coproporphyrinogen oxidase (Cpox nct mutant allele) [13]. The mutation in BALB.NCT-Cpox nct is a single amino acid substitution at the 380th position of the CPOX enzyme, from leucine to arginine (R380L). A patient with HCP heterozygous for the R391W amino acid substitution at the homologous position of the R380L substitution in BALB.NCT-Cpox nct was reported [1]. The mutant CPOX-R391W was approximately 22% as active as the wild type. Intriguingly, the reduced activity of the mutant CPOX-R380L of BALB.NCT-Cpox nct (~15% of wild-type) was consistent with the human CPOX-R391W [13]. Also, concurrent with the decrease in CPOX activity, BALB.NCT-Cpox nct had a drastic increase in coproporphyrin in the serum and liver, which persisted from a young age in both sexes. These results suggested that BALB.NCT-Cpox nct mice exhibit clinical symptoms of HCP. In this study, this possibility was investigated by biochemical, clinical, and histopathological analysis of BALB.NCT-Cpox nct .

Mice and ethical statement
Breeding pairs of inbred BALB.NCT-Cpox nct congenic mice [14] that carry the mutant Cpox nct gene in a homozygous state [13] were obtained from the RIKEN BRC (Tsukuba, Japan) (RBRC00422) via the National BioResource Project of MEXT/AMED (Tokyo, Japan) and subsequently bred by brother × sister mating at the Institute of Experimental Animals, Shinshu University. The BALB/c mice used as controls were purchased from Japan SLC, Inc. (Hamamatsu, Japan). These mice were maintained under clean conventional conditions at a temperature of 24 ± 2 • C under a light-controlled schedule (12-h light/dark cycle) with free access to a commercial diet (MF; Oriental Yeast Co. Ltd., Tokyo, Japan) and tap water. All experimental procedures involving mice were conducted following the Regulations for Animal Experimentation of Shinshu University and the ARRIVE guidelines. In addition, the animal protocol was reviewed by the Committee for Animal Experiments of Shinshu University and approved by the president of Shinshu University (approval number 019071).

Measurement of porphyrins and porphyrin precursors
Fresh urines were collected from 3-month-old male and female mice (n = 3) three times a day (morning, noon, and evening) for seven days and immediately frozen at − 80 • C. After thawed, the urines were centrifuged at 10,000 rpm for 10 min to remove insoluble components and samples from the same individuals were collected into one tube. Blood was drawn into a syringe with 10% EDTA-2K solution as an anticoagulant from the hearts of mice under isoflurane anesthesia. Measurements of porphyrins and porphyrin precursors were outsourced to the commercial laboratory BML, Inc. (Tokyo, Japan). Uroporphyrin and coproporphyrin contents in the urine and blood and protoporphyrin contents in the blood were measured with an L-6200 HPLC system (Hitachi High-Tech, Tokyo, Japan). Porphobilinogen and δ-aminolevulinic acid (δ-ALA) in the urine was measured by a spectrophotometer (7011, Hitachi High-Tech) and a UFLC (Shimadzu, Kyoto, Japan), respectively.

Heart rate and blood pressure measurement
All measurements were performed during the circadian cycle's light phase. The heart rate and blood pressure of 3-month-old mice were measured using an oscillometric technique and a computerized tail-cuff system (BP-98A-L, Softron Ltd., Tokyo, Japan). Each mouse was measured three times, and the mean was used as the measured value for each mouse.

Forelimb grip strength test
The tail of the 3-month-old mouse that gripped a bar connected to a digital force gauge (DS2-2N, IMADA, Toyohashi, Japan) was gently pulled backward, and the maximal grip force applied until the mouse released the bar was recorded. Each mouse was measured five times, with at least one minute separating each measurement, and the average of these measurements was used as the mouse's measured value.

Rotarod test
The motor coordination and balance of 3-month-old mice were analyzed using a rotarod apparatus (O'hara & Co., LTD, Tokyo, Japan). The device was programmed to accelerate the rotating speed of the rod from 4 to 40 rpm in 300 s. It was determined how long each mouse could remain on the rotating rods. Measurement was repeated three times with trials at least 15-min intertrial intervals, and the meantime was recorded as the duration observed for each mouse.

Histological examination
After being sacrificed by cardiac puncture under deep isoflurane anesthesia, the liver and skin from the dorsal neck, midback, and buttocks were removed from mice, formalin-fixed, paraffin-embedded, and sliced to a thickness of 4 μm for histological analysis. For the Masson-Goldner staining of liver sections, the Masson-Goldner staining kit (100485, Merck, Darmstadt, Germany) and Weigert's iron hematoxylin kit (115973, Merck) were used. After hematoxylin-eosin (HE) staining and Masson-Goldner staining, sections were viewed using an optical microscope. For transmission electron microscopy (TEM), 1-2 mm pieces of the liver were prepared, prefixed in 2.5% glutaraldehyde, and postfixed in 1% osmium tetroxide, followed by embedding in epoxy resin. The specimens were examined using a JEM-1400 electron microscope (high contrast).

Immunoblot analysis
Liver samples were homogenized in ice-cold RIPA buffer (Santa Cruz Biotechnology, Dallas, TX, USA). The homogenates were centrifuged for 15 min at 4 • C and 13,000 rpm, and the pellets were resuspended in RIPA buffer. The protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific). An aliquot equivalent to 8-10 μg protein was separated by SDS-PAGE using Tris-Tricine-SDS buffer and 8% polyacrylamide gels. The separated proteins were then transferred to a polyvinylidene difluoride membrane (Immobilon, 0.2 μm pore, (Merck Millipore, Burlington, MA, USA)) and incubated overnight at 4 • C with anti-lamin A/C (E-1) mouse monoclonal antibody (1:500, sc-376248, Santa Cruz Biotechnology) or anti-cytokeratin 8 rabbit monoclonal antibody (1:7500, ab53280, Abcam). The target proteins were detected by enhanced chemiluminescence after the membranes were then incubated with horseradish peroxidaseconjugated anti-rabbit IgG (1:4000, 7074S, Cell Signaling Technology, Danvers, MA, USA) or anti-mouse IgG (1:4000, 7076S, Cell Signaling Technology) for 1 h at room temperature.

Hematological and blood biochemistry tests
Under isoflurane anesthesia, blood samples were drawn from the hearts of mice into a syringe with or without 10% EDTA-2K solution as an anticoagulant. The hematological parameters were determined with an automated cell counter (XT-2000iV, Sysmex Co., Ltd., Kobe, Japan). Plasma or serum was obtained after 30 min of centrifugation at 3,000 rpm. Using the Mouse Ferritin ELISA Kit (ab157713; Abcam) and a microplate reader (SpectraMax M5), plasma ferritin levels were determined. Serum aspartate aminotransferase, alanine aminotransferase, and total bilirubin levels were determined using L-type Wako AST・J2, L-type Wako ALT・J2 (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), and Nascauto VL T-BIL (Alfresa Pharma, Ohsaka, Japan), respectively and an auto-analyzer (Model 7180, Hitachi, Tokyo, Japan).

Statistical analysis
Results are expressed as mean ± SD. Two-tailed Student's t-test was employed for statistical analysis. P-value < 0.05 was considered statistically significant.
The blood coproporphyrin contents were significantly higher in males than in females. Female but not male BALB.NCT-Cpox nct had significantly higher blood protoporphyrin contents than sex-matched BALB/c. Blood uroporphyrin contents were <1 μg/dL RBC in both BALB.NCT-Cpox nct and BALB/c of both sexes. Urinary coproporphyrin contents of male and female BALB.NCT-Cpox nct were 385-and 219-fold higher, respectively than that of sex-matched BALB/c (Table 1). Also, urinary δ-ALA, porphobilinogen, and uroporphyrin contents were significantly elevated in BALB.NCT-Cpox nct compared to BALB/c in both sexes. Urinary contents of these porphyrins and porphyrin precursors of female BALB.NCT-Cpox nct were significantly lower than those of males. The color of BALB.NCT-Cpox nct urine appeared reddish (Fig. 1). When irradiated with a black light (peak wavelength of 375 nm), the urine and feces of BALB.NCT-Cpox nct emitted a red fluorescence suggestive of excessive porphyrins in the feces as well.

BALB.NCT-Cpox nct mice manifest neuromuscular symptoms
The heart rate, blood pressure, grip strength, and rotarod performance of 3-month-old mice are summarized in Table 2. All data are expressed as mean ± SD (n = 3). **P < 0.01, and ***P < 0.001 vs. BALB/c (Student's t-test). † P < 0.05, and † † P < 0.01 vs. male BALB.NCT-Cpox nct (Student's t-test). The heart rate of BALB.NCT-Cpox nct was like that of BALB/c of the same age and sex. Female BALB.NCT-Cpox nct had significantly higher systolic blood pressure than female BALB/c (+6.4 mmHg; P < 0.05). In contrast, male BALB.NCT-Cpox nct had a systolic blood pressure comparable to male BALB/c. The diastolic blood pressure of BALB.NCT-Cpox nct was comparable to that of BALB/c of the same age and sex. Forelimb grip strength of female and male BALB.NCT-Cpox nct was 63% (P < 0.001) and 55% (P < 0.001) of age-and gender-matched BALB/c, respectively. Rotarod performance of BALB.NCT-Cpox nct was significantly lower than that of age-and gender-matched BALB/c, indicating that BALB.NCT-Cpox nct had impaired motor coordination.

Male BALB.NCT-Cpox nct mice exhibit NASH-like pathology in the liver
Under a light microscope, male BALB.NCT-Cpox nct liver exhibited severe histopathologic changes, but not female ones. In the pericentral and midlobular regions of 3-month-old males, hematoxylin-eosin (HE) staining revealed fatty changes, hepatocyte hypertrophy, and karyomegaly ( Fig. 2A). These conditions expanded gradually with age to the whole lobules of liver ( Supplementary Fig. S1). Also observed were nuclear pseudoinclusions (invaginations of cytoplasm into the nucleus) in hepatocytes. Transmission electron microscopy demonstrated the expansion and deformation of the hepatocyte nucleus (Fig. 2B). Massive pigment-filled Kupffer cell aggregates were also observed in male BALB. NCT-Cpox nct . This transformation was most noticeable in the pericentral regions ( Fig. 2A).
Mallory-Denk bodies (MDBs), a kind of hepatocellular inclusions, play an important role as histological and potential progression markers in several liver diseases, including alcoholic and nonalcoholic steatohepatitis, hepatocellular carcinoma, chronic cholestasis [15,16]. We employed a sensitive double immunoflurescence method with antikeratin 8 and anti-p62 antibodies to detect MDBs [17] and revealed the presence of MDBs in the liver of male BALB.NCT-Cpox nct mice. MDBs were only observed sporadically in the liver of 3-9-month-old BALB. NCT-Cpox nct but were abundant in 12-18-month-old ones ( Fig. 2C and Supplementary Fig. S2). Masson-Goldner staining of the liver revealed perivenular fibrosis in male BALB.NCT-Cpox nct (Fig. 2D). The perivenular fibrosis appeared at three months of age and progressed with age, with the formation of fibrosis bridging beginning at nine months of age ( Supplementary Fig. S3). These histopathological changes were not observed in 1-month-old mice but were observed in all males examined older than three months (Table 3). Additionally, one in nine males aged nine months and four of nine males aged 12-18 months had liver tumors (Fig. 2E). In tumor sections, numerous cells were positive for the proliferation marker Ki-67. These histopathological alterations resembled those observed in NASH. Immunoblot analysis revealed aggregates and polymerized forms of intermediate filament proteins lamin A/C and keratin 8, in the liver of male BALB.NCT-Cpox nct (Fig. 2F). Consistent with the histopathological and biochemical findings, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels as indicators of liver injury were significantly elevated in male BALB.NCT-Cpox nct compared to male BALB/c after three, and six months of age, respectively, whereas those in female BALB.NCT-Cpox nct mice at ages 3 and 9 months were comparable to those of age-and sex-matched BALB/c mice ( Table 3). The total serum bilirubin level of BALB.NCT-Cpox nct mice fell within the normal range, indicating that they did not suffer from cholestasis.

Male BALB.NCT-Cpox nct mice exhibit sclerodermatous skin pathology
The subcutaneous fatty tissue of the HE-stained male BALB.NCT-Cpox nct skin was often discernible and extremely thin (Fig. 3). Also, slight thickening and fibrosis of the dermis were observed. Some males older than three months exhibited these sclerodermatous changes, whereas no such alterations were observed in females (Table 4). However, the extent of these changes was highly variable between individuals and regions of skin on the same individual (Supplementary Fig. S4 and Supplementary Table S1).

BALB.NCT-Cpox nct mice exhibit microcytic anemia
The data in Table 5 pertain to the hematology of the peripheral blood of 3-month-old mice. BALB.NCT-Cpox nct had significantly lower levels of hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), and mean corpuscular hemoglobin (MCH) versus BALB/c matched for age and gender. In addition, the coefficient of variation for RBC distribution width (RDW-CV) was significantly greater in BALB. NCT-Cpox nct compared to BALB/c. The plasma ferritin levels of male BALB.NCT-Cpox nct were significantly higher compared to male BALB/c, whereas no significant strain differences were observed for the female ferritin levels. These results indicated that BALB.NCT-Cpox nct had microcytic anemia.

Discussion
In this study, we demonstrated that BALB.NCT-Cpox nct mice carrying a hypomorphic mutation in the Cpox gene presented HCP symptoms. Most notably, the symptoms included NASH-like liver pathology and sclerodermatous skin pathology. Curiously, however, these symptoms were observed only in males. Overall, the BALB.NCT-Cpox nct showed both similarities and differences with HCP patients (Table 6). Additionally, we discovered that BALB.NCT-Cpox nct had microcytic anemia.
To the best of our knowledge, BALB.NCT-Cpox nct is the only model mouse that spontaneously manifests severe coproporphyria phenotypes. BALB.NCT-Cpox nct mice exhibit the phenotypes supposedly because CPOX activity in the mice is sufficiently low, and a high coproporphyrin level persists systemically from a young age [13]. Presumably, the remaining CPOX activity in BALB.NCT-Cpox nct mice (around 15% of wild-type) [13] would be sufficient to prevent embryonic death and permit postnatal growth and reproduction. However, even under normal physiological conditions, this CPOX activity would be insufficient for the conversion of coproporphyrinogen III to protoporphyrinogen IX, resulting in the systemic accumulation of coproporphyrinogen. An HCP patient due to a homozygous arginine-to-tryptophan substitution (R231W) mutation of CPOX was reported [18,19]. The onset of HCP symptoms in this patient was unusually early in childhood. She exhibited hypertrichosis and skin pigmentation affecting the face and dorsum of both hands when she was four years old. At 10 years, she was admitted to the hospital for persistent vomiting, abdominal pain, and constipation, and diagnosed with HCP. The lymphocyte CPOX activity of the patient was only 2% of normal. The patient's urinary levels of δ-ALA, All data are expressed as mean ± SD (n = 6). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. BALB/c (Student's t-test). porphobilinogen, and coproporphyrin were 19, 36, and 151 times higher than the normal mean or upper normal limits [18,20]. Thus, although the position of amino acid substitution in CPOX is different, the pathophysiological condition of BALB.NCT-Cpox nct mice appears comparable to that of this HCP patient. The most notable finding in this study was that male BALB.NCT-Cpox nct presented NASH-like pathology and tumor formation in the liver. As far as we know, there have been no reports of NASH-like changes in HCP patients. HCP is clinically similar to other acute hepatic porphyrias: intermittent acute porphyria, variegate porphyria, and δ-aminolevulinic acid dehydratase porphyria, which are caused by mutations in the genes for porphobilinogen deaminase, protoporphyrinogen oxidase, and δ-aminolevulinic acid dehydratase, respectively. The risk of primary liver cancer in patients with acute hepatic porphyrias has been confirmed (incidence = 1.5-35%) [6][7][8][9][10][21][22][23][24]. Similarly in mice, protoporphyrinogen oxidase (PPO)-inhibiting herbicides cause accumulation of protoporphyrin IX in tissues, especially the liver. Accordingly, long-term administration of these herbicides to mice induces hepatocellular adenoma and carcinomas [25][26][27][28][29].

Cpox nct
With regards to the mechanisms for the development of liver lesions in male BALB.NCT-Cpox nct , immunoblot analysis of the liver revealed aggregates and polymerized forms of cellular proteins in males. It has been demonstrated that protein aggregations of the nuclear intermediate filament (IF) protein (lamin) and the cytoplasmic IF protein (keratin 8) are formed in protoporphyrinogen IX-mediated liver injury [30,31]. In addition, the pellet fractions of HepG2 cell-free extracts treated with coproporphyrin have the potential to mediate protein aggregation [30]. ALT, AST, and total bilirubin data are expressed as mean ± SD (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. BALB/c (Student's t-test).    Table S1). All data are expressed as mean ± SD (n = 6). **P < 0.01, and ***P < 0.001 vs. BALB/c (Student's t-test). WBC, white blood cell; RBC, red blood cell; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW-CV, red blood cell distribution width -coefficient of variation; PLT, platelet.
IF proteins, belonging to major cytoskeletal protein families, serve important roles in the maintenance of structural integrity and cellular homeostasis [32,33]. The nuclear lamin IFs, as the main component of the nuclear lamina, play major roles in maintaining structural integrity, gene transcription, and protein transportation [34,35]. Keratin 8 forms IFs, and not only maintains cellular structural integrity but also functions in signal transduction and cellular differentiation. It has been demonstrated that coproporphyrin binds proteins possibly through the deprotonation of the carboxylate moiety of the propionic acid side chain, and leads, in the presence of oxygen, to protein oxidation and aggregation in the liver of these porphyria model mice [9,30,31,36]. Thus, the accumulation of protein aggregates and polymers in the cells would disturb cellular morphology and function, and lead to the development of various pathological conditions in the liver of male BALB.NCT-Cpox nct . To clarify the role of accumulated coproporphyrin and/or porphyrin precursors in developing hepatic pathology, it is necessary to conduct additional research on BALB.NCT-Cpox nct .
In the skin of male BALB.NCT-Cpox nct , sclerodermatous pathology was also observed. According to our knowledge, scleroderma complications in HCP patients have not been reported. Some patients with porphyria cutanea tarda, caused by mutations in the uroporphyrinogen decarboxylase gene, exhibit similar photosensitivity, and fragility, leading to repeated blistering and fissuring that progresses to fibrosis and becoming sclerodermatous in the late stages [37].
Curiously, the manifestation of the NASH-like and sclerodermatous pathologies was restricted to males of BALB.NCT-Cpox nct . The reason for the absence of the pathologies in female BALB.NCT-Cpox nct is not clear. Intriguingly, urinary levels of δ-ALA, porphobilinogen, uroporphyrin, and coproporphyrin of female BALB.NCT-Cpox nct were considerably lower than those of males (Table 1). These levels might be insufficient to develop pathological changes. Further studies would be required to determine whether this is the reason for the absence of symptoms in females as well as females have lower porphyrin precursor levels despite having the identical Cpox nct mutation. The elucidation of the mechanisms responsible for the suppression of hepatic and cutaneous pathologies in female BALB.NCT-Cpox nct should lead to the development of new effective therapies for HCP. In contrast, both female and male BALB.NCT-Cpox nct mice exhibited a decline in forelimb grip strength and deterioration of motor coordination. The pathogenesis of these neuromuscular symptoms may differ from that of liver and skin pathogenesis. Neurovisceral symptoms of hepatic porphyrias are considered to be related to the accumulation of δ-ALA [38,39]. The urine δ-ALA contents in female BALB.NCT-Cpox nct was 9.2 mg/L, which was equivalent to the levels in symptomatic acute intermittent porphyria patients [40]. This may be the reason why female BALB.NCT-Cpox nct also presented neuromuscular symptoms.
Both sexes of BALB.NCT-Cpox nct mice spontaneously developed microcytic anemia. The magnitude of hematological parameter changes was comparable to that of heterozygous Cpox mutant mice [11,12]. Male but not female BALB.NCT-Cpox nct had elevated peripheral blood ferritin levels. The high blood ferritin levels of male BALB.NCT-Cpox nct may result from the release of ferritin from damaged hepatocytes [41].

Declaration of Competing Interest
The authors have no conflict of interest.

Data availability
Data will be made available on request.